豚鼠气单胞菌CB101中几丁质酶的纯化、鉴定及其基因克隆

豚鼠气单胞菌CB101在胶体几丁质诱导下产生多种分子量不同的几丁质酶,其中分子量最大的一个几丁质酶Chi1(约91.5kDa)的编码基因已经被克隆.本文报道从CB101发酵液经硫酸铵盐析,DEAE-纤维素柱层析和MonoQ离子交换柱层析,分离纯化出其中的一个分子量约为60KDa的几丁质酶.N端测序的结果表明该酶是Chi1剪缺掉N末端信号肽部分、C末端A区和两个几丁结合区的产物.构建了CB101基因组的cosm id文库,从中筛选到9个产几丁质酶的克隆子,克隆子分析表明它们包含的几丁质酶基因都是chi1.蛋白分离、纯化和基因克隆的实验都暗示CB101的几丁质酶系统只包含一个几丁质酶基因.

Purification ,characterization and gene clone of Chitinase from Aeromonas caviae CB101

Aeromonas caviae CB101 can secrete multiple chitinases into the culture supernatant as revealed by chitinase activity staining.One gene chi1 has been cloned and sequenced.It encodes a protein of 865 amino acids with a signal peptide at the N-terminal,of which a polycystic-kidney-disease(PKD)-like domain,a triosephosphate-isomerase(TIM) catalytic region,a receptor-for egg jelly(REJ)-like domain and two chitin-binding-domain(chBDs) locate from the N-terminal to C-terminal.A chitinase was isolated from the culture and purified by the steps of ammonium sulfate precipitation,DEAE-Sepharose and Mono Q anion exchange column chromatography.The result of it's N-terminal sequencing approves that it is the truncation of the signal peptide and some C-terminal domains of the cloned chitinase Chi1.When spray the CB101's cosmid library on the plates containing the antibiotic and 1% colloid chitin,nine clones that can utilize chitin were gotten and were proved to be chitinase.The isolation and purification of chitinase following N-terminal sequencing and other experiments suggest that the Aeromonas caviae CB101 can only secrete one chitinase,which are Chi1 and its truncates.